Coherent Raman scattering (CRS) imaging is based on a multiphoton scattering process that employs two near-infrared laser pulses to excite Raman modes in the midinfrared spectral range. Owing to its chemical selectivity without labelling, it has found a wide scope of applications in biomedical microscopy, such as live cell, tissue, and DNA imaging, over the past years. Its most prominent representatives are coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS). In both cases, two beams, the so-called pump and Stokes beam, with an energy difference matching the Raman resonance, interact with each, giving rise to the generation of a new frequency (CARS) or to an energy exchange between the two beams (SRS).
Here, an ultra-low noise and fully automated laser system is developed for CRS microscopy. Based on solid-state femtosecond technology, combined with optical parametric frequency conversion this multicolor system reaches the shot noise limit at modulation frequencies of 1 MHz and above. Delivering tunable radiation in the 750 – 950 nm and 1.4 - 2.0 μm ranges, together with a spectrally fixed beam at 1043 nm, it is perfectly suited for coherent Raman microscopy in the range of 1015 – 3695 cm-1, as well as for multiphoton excitation microscopy. With customizable pulse durations from hundred femtoseconds to picoseconds, efficient excitation and spectral resolution down to 13 cm-1 are possible. All three output beams are inherently synchronized and, in addition, the Stokes and pump beams are spatiotemporally overlapped with a precisely controllable temporal delay. The unique robust frequency conversion design requires no active stabilization electronics, which usually affect the system stability, noise, and handling in a negative manner. The presented system is fully automated and allows wavelength tuning with subnanometer precision via its hardware control panel or by remote access.
In order to evaluate the system performance, i.e., the tuning width and resolution, both the spectrally very broad SRS response from a D2O and H2O mixture are recorded, as well as the narrow response of acetone. To demonstrate the capability of the presented light source with respect to imaging applications a mixture of micrometer sized Polystyrene (PS) and PMMA is investigated in a basic confocal scanning microscope setup. Within this proof-of-concept, it can be shown that the system allows chemical selective imaging with video frame rates. Related research results were published in Advanced Photonics Volume 1 Issue 5 (Heiko Linnenbank, Tobias Steinle, Florian Mörz, Moritz Flöss, Han Cui, Andrew Glidle, Harald Giessen. Robust and rapidly tunable light source for SRS/CARS microscopy with low-intensity noise).
(a) Schematic experimental setup. An Yb-solid-state oscillator allows synchronous generation of the Raman Stokes and pump beams. Using an etalon, the narrowband Stokes beam with picosecond pulse duration is obtained, whereas the Raman pump beam is generated by pumping an OPO, which is subsequently frequency doubled in a periodically poled crystal employing the effect of spectral compression. (b) 94μm× 94 μm raster scan images of 6-μm diameter polymer beads. The contrast mechanism is the scattering by the beads, which is lowering the DC-value of the detected Stokes beam. (c) SRS signal images from the raster scan in (a), recorded or a Stokes-pump energy difference of 3067 cm-1 (PS-band) and 2953 cm-1 (PMMA-band), revealing two different kinds of beads and thus presenting the chemical selectivity.
